The overall aim of this project is to characterize the mechanisms by which a rhodopsin molecule excites the visual receptor. In the present proposal there are three major objectives: (1) Recent work in this project has demonstrated that low concentrations of oxidizing agents can rapidly close the light-regulated cation channels ("PL") in isolated rod outer segments (ROS), and that exceedingly low concentrations of reducing agents, as well as membrane impermeant sulfhydryl binding reagents, can rapidly open cation channels ("PSH") in the isolated ROS. With the evidence available to date, the SH-sensitive permeability, PSH, is indistinguishable from the light-regulated permeability, PL, so perhaps both represent the same cation channel. Thus, the first aim of this project is to isolate and characterize the molecules in the ROS plasma membrane that contain these highly sensitive SH groups. The binding of less than 10,000 SH reagents/ROS opens PSH. This is such a small #/ROS that in addition to radioactive labels, two new labeling techniques will be tried: colloidal gold particles (individually visible in EM), and/or fluorescently labeled methacrylate microspheres (individually visible in fluorescence LM) will be functionalized with SH seeking reagents and then reacted with isolated ROS to determine rates of reaction, total number/ROS, and spatial distribution of these SH sites. Patch-clamp methods will be used to attempt to purify, identify, and characterize the channels responsible for PL and PSH. (2) In addition to studying these SH-containing molecules, attempts will be made to identify other molecular sites that are highly susceptible to oxidation upon isolation of the ROS. It seems likely that some of the degradative diseases of photoreceptors may result from diminished protection or replacement of these "most labile" sites. The rates of both lipid peroxidation and SH oxidation will be measured in isolated ROS exposed to various concentrations of oxidizers and compared with the rates at which the oxidizers close the PL channels. (3) As in previous years of this project, the role of diffusion in setting the timecourse of visual excitation will continue to be investigated. The effects of experimentally inducing changes in the viscosities of the disc membrane and the cytoplasm will be evaluated by monitoring both the receptor potential and the concurrent light-scattering signals. Fluorescence photobleaching and recovery techniques will be used to quantitate the changes induced in the viscosities.